In vivo Drug Screening to Identify Anti-metastatic Drugs in Twist1a-ERT2 Transgenic Zebrafish

Here, we present an in vivo drug screening protocol using a zebrafish model of metastasis for the identification of anti-metastatic drugs. A tamoxifen-controllable Twist1a-ERT2 transgenic zebrafish line was established to serve as a platform for the identification. By crossing Twist1a-ERT2 with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish show spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of epithelial to mesenchymal transition (EMT). This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the identification of anti-metastatic drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test drug on metastasis in five days, by comparing the frequencies of the fish showing abdominal and distant dissemination patterns in the test drug–treated group with those in the vehicle-treated group. Our study previously identified that adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), has a suppressor effect on cell dissemination in the model. Furthermore, we validated that a pharmacologic and genetic inhibition of HSD11β1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Taken together, this protocol opens new routes for the identification of anti-metastatic drugs. Graphical overview Timing Day 0: Zebrafish spawning Day 8: Primary tumor induction Day 11: Chemical treatment Day 11.5: Metastatic dissemination induction in the presence of a test chemical Day 16: Data analysis


Background
Metastasis is responsible for approximately 90% of cancer-associated mortality. It proceeds through multiple steps: invasion, intravasation, survival in the circulatory system, extravasation, colonization, and metastatic tumor formation in secondary organs with angiogenesis (Nguyen et al., 2009;Chaffer and Weinberg, 2011;Welch and Hurst, 2019). The dissemination of cancer cells is an initial step of metastasis, and its molecular mechanism involves a local breakdown of basement membrane, loss of cell polarity, and induction of epithelial to mesenchymal transition (EMT). EMT plays a central role in early embryonic morphogenesis; its process enables various types of epithelial cells to convert into mesenchymal cells, through a downregulation of epithelial markers such as E-cadherin and an upregulation of mesenchymal markers such as vimentin. Twist, a basic helix-loop-helix transcription factor, plays a critical role in inducing the EMT program (Tsai and Yang, 2013;Lu and Kang, 2019). Past studies showed that elevated expression of Twist is associated with poor survival rates in patients with cancer; also, ectopic expression of Twist confers metastatic properties on cancer cells through induction of EMT (Yang et al., 2004;Tsai et al., 2012). Cancer research using zebrafish as a model has attracted attention because this model offers many unique advantages that are not readily provided by other animal models (White et al., 2013;Osmani and Goetz, 2019). Furthermore, the zebrafish system has also been increasingly recognized as a chemical screening platform because it provides the advantage of high-throughput screening in an in vivo vertebrate setting with physiologic relevance to humans (Zon and Peterson, 2005;Letrado et al., 2018;Nakayama et al., 2021bNakayama et al., , 2022aNakayama et al., and 2022b. Our study previously established a tamoxifen-controllable Twist1a-ER T2 transgenic zebrafish line that serves as an in vivo drug screening platform for the identification of anti-metastasis drugs targeting metastatic dissemination of cancer cells. By crossing Twist1a-ER T2 with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish showed spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of an EMT Lu et al., 2021). The dissemination patterns are generally divided into three categories: (i) local dissemination, in which disseminated mCherry-positive cells exist in close proximity to the liver; (ii) abdominal dissemination, in which the cells spread throughout the abdomen; and (iii) distant dissemination, in which the cells are observed over a broad region from the trunk to the tail ( Figure 1A). This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the discovery of anti-metastasis drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test chemical through comparing the frequencies of the fish showing the abdominal and distant dissemination patterns in the test drug-treated group with those in the vehicle-treated group. Previous studies confirmed that ki16425 (a LPA1 inhibitor) or Y27632 (an inhibitor of Rho-associated coiled-coil-containing protein kinase), which have been reported to suppress metastasis in mice models of metastasis (Itoh et al., 1999;Boucharaba et al., 2006), could suppress cell dissemination in the fish model. In vivo drug screen using this model identified adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), as having a potential to suppress metastatic dissemination of cancer cells ( Figure 1B and 1C). Furthermore, pharmacologic and genetic inhibition of HSD11β1 were validated to suppress metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model 2021a). Taken together, our model offers an in vivo drug screening platform for the identification of anti-metastatic drugs.   (Westerfield, 2007).
d. Feeding of the zebrafish larvae began at 5 dpf. A pinch of micron powder was diluted in 1 mL of E3 medium and added to the zebrafish larvae every morning. The zebrafish larvae were transferred into each well of a 6-well plate at a time point between 8 and 11 dpf, and a few drops of the E3 medium were added to each well every morning.

B. Collect Twist1a-ER T2 /xmrk double transgenic zebrafish (Day 6)
1. Transfer the zebrafish larvae into a 50 mL tube. 2. To anesthetize the fish, add 2% (v/v) phenoxyethanol to the E3 medium with the micron powder that contains the fish. Thus, the final concentration of phenoxyethanol is 0.02%. 3. Array the fish on a lid of a 150 mm plastic dish. 4. Visualize under a fluorescence microscope and use a pipette to collect the fish expressing green fluorescent protein (GFP) in the skin (Figure 2). images of GFP and mCherry signals in Twist1a-ER T2 /xmrk double transgenic zebrafish at 6 days postfertilization (dpf). Scale bar, 200 μm.

Figure 2. A Twist1a-ER T2 /xmrk double transgenic zebrafish expresses green fluorescent protein (GFP) in a skin-specific manner (A) and mCherry in a liver-specific manner (B). Representative
5. Among the GFP-positive fish, visualize under a fluorescence microscope and use a pipette to collect those expressing mCherry in the liver (Figure 2). 6. Transfer the collected fish into a 150 mm plastic dish containing E3 medium. 7. Maintain the fish for two days in an incubator set at 27 °C. Notes: a. At 6 dpf, Tg (fabp10a:mCherry-T2A-Twist1a-ER T2 ) begins to express the gene coding mCherry-T2A-Twist1a-ER T2 in a liver-specific manner. Tg (fabp10a:TA; TRE:xmrk; krt4:GFP) expresses GFP in a skinspecific manner. Therefore, Twist1a-ER T2 /xmrk double transgenic zebrafish is indicated as mCherry-and GFP-positive in the liver and skin, respectively. b. The screening process is divided into two steps. Firstly, zebrafish possessing the xmrk transgene are screened at 3-5 dpf

D. Chemical treatment (Day 11)
1. Aliquot approximately 20 zebrafish larvae into each well of a 6-well plate with 8 mL of E3 medium containing doxycycline (30 μg/mL) at a time point between 8 and 11 dpf. 2. Add each test chemical to each well of the plate at a final concentration of 5 μmol/L. Notes: a. All test chemicals are dissolved in DMSO at a concentration of 10 mmol/L (stock solution). b. 4 μL of each stock solution (10 mmol/L) is diluted with 100 μL of E3 medium and added into each well of the plate containing 8 mL of E3 medium.

E. Metastatic dissemination induction in the presence of a chemical (Day 11.5)
1. Beginning 12 h after the addition of the test chemical, treat the fish with 0.1 μmol/L of 4-OHT in E3 medium for five days. 2. Change the E3 medium containing doxycycline, 4-OHT, and the test chemical every two days. Notes: a. The quality of the E3 medium is critical for the survival of the zebrafish larvae. The fish excrete waste materials including urine. Waste materials and leftover foods are harmful for the viability of the fish. Thus, the E3 medium should be changed before a high concentration of waste materials is reached. b. We recommend changing the E3 medium every two days after 8 dpf.

Limitations
There is a limitation on the number of Twist1a-ER T2 /xmrk double transgenic zebrafish that can be prepared (a few hundred). This limitation determines how many chemicals can be tested in one screening session. To test one chemical, approximately 20 double transgenic fish are needed. Following Mendel's laws, the rate of the double transgenic fish production is 25% when a heterozygous Twist1a-ER T2 transgenic zebrafish is crossed with a heterozygous xmrk transgenic zebrafish. If 20 chemicals are to be tested, at least 400 double transgenic fish would be required. To prepare 400 double transgenic fish, pairs of Twist1a-ER T2 and xmrk fish would need to generate approximately 2,000 embryos. Thus, when following the protocol described above, a maximum of 20 test chemicals can be evaluated.